The proposed research is designed to elucidate the molecular interactions which are responsible for the specific transcription of an isolated eukaryotic gene, by the homologous RNA polymerase, in vitro. The system which will be studied is the transcription of the ribosomal RNA gene from Tetrahymena pyriformis by its form I RNA polymerase. The in vivo synthesis and processing of the precursor rRNA molecule will be studied and the nucleotide sequence of its 5' and 3' termini will be determined. Methods for the large scale isolation of the ribosomal DNA, either as free DNA or as a deoxyribonucleoprotein complex will be developed. The form I RNA polymerase will be purified to homogeneity and its activity on both the protein free rDNA and on the rDNA-nucleoprotein complex will be studied. The nucleotide sequence of the 5' and 3' termini of the in vitro synthesized RNA will be determined and will be compared to the termini of the in vivo synthesized precursor rRNA molecule. The nucleotide sequence analysis will provide an unambiguous assay for specific transcription in vitro since the 5' and 3' ends of the RNA are transcribed from the sites of initiation and termination of transcription. Through an analysis of the RNA transcribed in vitro either from pure rDNA or from the rDNA nucleoprotein complex, it will be possible to assess the roles of chromosomal proteins in determining transcription specificity.